Journal of AOAC International最新文献

Background: Batches of Pigment Red 4 (PR4) submitted to the U.S. FDA for certification as D&C Red No. 36 (R36) must comply with specifications listed in the U.S. Code of Federal Regulations (CFR). Currently, a tedious TLC method is used to enforce limiting specifications for four subsidiary colors: Pigment Orange 5 (PO5, CAS 3468-63-1) and Yellow-1-Naphthol (Y1N, CAS 36265-89-1), each ≤ 0.5%, and Pigment Red 1 (PR1, CAS 6410-10-2) and Pigment Red 6 (PR6, CAS 6410-13-5), each ≤ 0.3%.

Objective: To develop improved analytical methods for determination of the subsidiary colors in samples from batches submitted for certification as R36.

Methods: HPLC and UHPLC were the techniques used for development of the new methods. Reference materials for PO5, PR1, and PR6 were purchased, and a standard for Y1N was synthesized, with its identity confirmed by HRMS and NMR. Calibration curves were prepared for quantification of the subsidiary colors by each method.

Results: Both newly developed methods enabled baseline separation of the four subsidiary colors and their identification based on defined retention times, elution sequence, and UV/Vis spectra. Linearity was demonstrated for both methods with R2 values > 0.999 for each of the analytes. Ranges of other validation data included: LOD, 0.006-0.018%; LOQ, 0.007-0.05%; recoveries, 85.5 ± 7.4% - 101.8 ± 2.0%.The new methods were implemented to quantify the analytes in 24 surveyed batches of R36. They yielded identical or nearly identical values for the analyzed subsidiary colors. Obtained levels in all but two samples were below CFR limits.

Conclusions: For routine batch-certification analyses of subsidiary colors in R36, either the HPLC or UHPLC method developed in this study can replace the outdated TLC procedure.

Highlights: The R36 subsidiary colors PO5, Y1N, PR1, and PR6 can be accurately quantified using the newly developed HPLC and UHPLC methods.

Background: Carbamates have been used in intentional poisoning cases to cause death in both humans and animals. Granulated material containing carbamate pesticides is often applied to the victim's food, water, or drink. Toxicological analysis is essential to confirm such cases.

Objective: This study aims to identify and quantify carbamates (methomyl, aldicarb, propoxur, carbofuran, and carbaryl) present in granular formulations used to adulterate food, through the application of chromatographic and vibrational microspectroscopic techniques.

Methods: Thin layer chromatography (TLC), liquid chromatography with diode array detector (LC-DAD), gas chromatography-mass spectrometry (GC-MS), Attenuated total reflectance Fourier transform infrared (ATR-FTIR), and Raman microspectroscopy were performed. An aliquot of granular formulation was manually separated for chromatographic methods; dilutions were employed as test material treatment, while vibrational microspectroscopy techniques used raw material for toxicological analysis. A material similar to a shredded sausage containing suspected granular formulation was evaluated in chromatographic tests, and carbofuran was determined.

Results: The proposed TLC methodology for identifying carbamates proved unfeasible due to insufficient chromatographic separation. The LC-DAD method was fully validated with success for methomyl, aldicarb, propoxur, carbofuran, and carbaryl, revealing a concentration of 744 µg/g of carbofuran in the suspected material, which was further confirmed by GC-MS. Raman microspectroscopy was not precise enough for carbamate identification, but ATR-FTIR demonstrated high specificity and robustness in identifying carbamate pesticides present in the test material.

Conclusion: This study allowed determining the profile of carbamate pesticides in granular material when present in adulterated food/feed. To isolate the granules manually reduces the carrying of matrix interferents from food and/or feed. Furthermore, the techniques applied require only the raw material or its diluted versions, avoiding steps, high consumption of organic solvents, and reducing analysis time and cost.

Highlights: LC-DAD was successfully validated for multiple carbamates. Minimal test material prep reduces solvent use, analysis time, and costs.

Background: Micro and nano plastics from textiles, rubber, cleaning products, etc. present in living and working environments can release toxic compounds, added to plastics to improve their properties.

Objective: This study explores the presence of plastic additives in settled dust from domestic and workplace indoor environments.

Methods: A Standard Reference Material was used to verify the validity of analytical method. Settled dust from two occupational environments (a sail loft and a tire shop), and from the home of a worker in the sail loft, is extracted, obtaining two fractions to be injected in GC-MS and in HPLC-MS/MS to detect and quantify 32 plastic additives. Results from working environments were also compared with those from a treating plant of waste electrical and electronic equipment (WEEE).

Results: After a clean-up procedure and the use of matrix-matched calibration curves, the method proved to be reliable.Significant differences among the concentrations of analytes extracted from the workplace settled dust were not found except for higher values of Bis-2-ethylhexyl adipate (DEHA) and Bis-2-ethylhexyl phthalate (DEHP) in the tire shop and of dimethyl phthalate (DMP) and Diisodecyl phthalate (DiDP) in the sail loft. Comparing the results from house and work environments, higher concentrations of additives were found at home. The results from the present workplaces compared with those from a WEEE treating plant showed that the latter was a much more polluted environment.

Conclusion: These preliminary results about the presence of plastic additives in the settled dust from living and working environments suggest that WEEE treating plant deserves more attention than others. Furthermore, the home environment hides some dangers for the presence of material that can release toxic compounds.

Highlights: Optimization of a method for detecting plastic additives in environmental settled dust.NIST SRM 2585 analysis of phthalates, adipates, phosphates, citrates, trimellitate, benzoate, sebacate, dicarboxylate, benzo-triazole, -triazine.Exposure to plastic additives occurs both in workplace and living environments.

Background: Pogostemon cablin (P. cablin) is a valuable medicinal plant used in traditional medicine and the fragrance industry, but quality control is challenging due to inconsistent stem-to-leaf ratios and frequent essential oil adulteration.

Objective: This study compares volatile components in different parts (aerial parts, stems, leaves) and essential oil of P. cablin to support quality control (not less than 20% leaf) and its rational use.

Methods: Volatile components in 21 batches of aerial parts, stems, leaves, and 13 batches of essential oils were analyzed using GC-MS. Multivariate curve resolution-alternating least squares (MCR-ALS) was used for resolving co-eluted peaks, and chemical fingerprinting with chemometric techniques like hierarchical cluster analysis (HCA), principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discrimination analysis (OPLS-DA) was applied.

Results: Volatile profiling identified 56, 47, 28, and 45 components in the aerial parts, leaves, stems, and essential oil of P. cablin, respectively. MCR-ALS resolved ten major volatile compounds to create chemical fingerprints for each analytical sample type. Quantitative analysis showed higher patchouli alcohol in leaves (12.47 mg/g) compared to stems (2.05 mg/g), while stems had more pogostone (2.71 mg/g vs. 1.40 mg/g in leaves). Aerial parts and essential oil showed significant compositional differences. Based on the results of qualitative and quantitative analysis, chemometric methods, including HCA, PCA, PLS-DA and OPLS-DA, clearly differentiated the four types of P. cablin analytical samples.

Conclusions: Significant differences in volatile components across P. cablin parts and its essential oil support quality control (not less than 20% leaf) and rational use.

Highlights: This study is the first to use MCR-ALS and other chemometrics for qualitative and quantitative analysis of P. cablin parts and essential oils, aiding quality control.

Background: Food contamination with mycotoxins has become a concern in recent years. Cyclopiazonic acid (CPA) is a mycotoxin found in foods, such as peanuts, corn, and cheese, which poses health hazards to humans.

Objective: To develop and validate an enzyme-linked immunosorbent assay (ELISA) for detecting CPA in various food matrices using appropriate pretreatment methods, with liquid chromatography/ultraviolet detection (LC/UV) and/or LC/time of flight mass spectrometry (TOF-MS).

Methods: Pretreatment of food was optimized using liquid-liquid extraction and solid-phase dispersive extraction (SPDE), and validated across liquid and solid foods. Indirect competitive ELISA was performed. The data from recovery experiments were analyzed using a one-way analysis of variance.

Results: All food specimens added at high concentrations yielded satisfactory ELISA results and exhibited a strong correlation with the LC/UV measurements. In the case of moderate-concentration additions, satisfactory accuracy was obtained for foods other than spices. At low concentrations, the results of ELISA and instrumental analyses (LC/UV and LC-TOF/MS) were comparable for sake, soy sauce, noodle soup, corn, and miso. However, elevated values were observed for the ELISA of cheese, peanuts, and spices.

Conclusion: Although the low recovery levels of CPA in spices suggests the need for a more effective cleanup method, the developed ELISA is suitable as a rapid screening method for CPA in various foods, with LC confirmation recommended in complex matrices.

Highlights: The use of SPDE for CPA analyses resulted in effective cleanup, which subsequently enabled the application of ELISA for screening.

Background: 5-Methytetrahydrofolate (5-MTHF), a reduced form of folate (vitamin B9) marketed as a potentially more bioavailable alternative to folic acid, is a key ingredient in dietary supplement (DS), particularly prenatal multivitamin/minerals. Its synthesis may produce racemic mixtures of bioactive L-5-MTHF and inactive D-5-MTHF, raising concerns about labeling accuracy, integrity, and health impacts. DS manufacturers consider D-5-MTHF an impurity and do not use racemic mixtures of 5-MTHF in their products.

Objective: This study aimed to develop and validate an HPLC-MS-based method to separate and quantify the D and L-5-MTHF diastereomers in various DS matrices and dosage forms.

Methods: A single-laboratory validated method was developed for determination of 5-MTHF isomers in different dosage forms of DS using targeted selected ion monitoring (Targeted SIM) with a ChiralPak HSA column. L-cysteine was selected as an extraction stabilizer, and the mobile phase was composed of 100 mM ammonium acetate and 1% v/v acetic acid in acetonitrile (ACN) at a 97:3 ratio.

Results: The HPLC/MS method was able to separate D-and L-5-MTHF diastereomers by isocratic elution within 20 min. It showed great linearity with the concentration range of 1 µg/mL to 160 µg/mL of 5-MTHF with R  2>0.99. The method was validated for the limit of detection (LOD), limit of quantitation (LOQ), linear range, specificity, and recovery according to the guidelines described Q2(R1) Validation of Analytical Procedures: Text and Methodology Guidance for Industry. The method was then applied for the analysis of 101 samples, and two samples were found to exceed the USP limit of ≤ 1% D-5-MTHF relative to L-5-MTHF (1.39% and 50.28%).

Conclusion: This performance of HPLC-MS method established effectively determines D- and L-5-MTHF ratios in complex DS, serving as a reliable tool to detect adulteration with racemic mixtures in prenatal and other 5-MTHF-labeled containing DS. It is significantly faster than previously reported method and possesses great selectivity in separation of the diastereomers in different form of DS.

Highlights: Successfully validated an HPLC-MS method for quantifying D- and L-5-MTHF diastereomers in diverse DS dosage forms, aiding enantiomeric purity assessment.

Background: Ivermectin (IVE) is an antiparasitic sold in the form of tablets, pastes, and injectable solutions. Neither the literature nor the official compendiums present an environmentally friendly method for analyzing the final IVE product by thin layer chromatography (TLC) assisted by digital images. This combination strengthens the advantages of cost, handling, time, execution and process optimization. Items, which the green and clean analytical chemistry values, and the National Environmental Methods Index (NEMI), Eco-Scale Assessment (ESA), Analytical GREEnness Metric (AGREE) and Green Analytical Procedure Index (GAPI) tools measure.

Objective: The objective of this work is to develop and validate an eco-efficient, fast, economical and easy-to-perform method for analysis of IVE injectable solution by TLC assisted by digital images.

Method: Silica gel plate, microsyringe and ethyl acetate: ethanol (13:2, v/v) as mobile phase were used in the method. The pixels were analyzed by Image J software after the spots were photographed under UV light.

Results: The method was selective when comparing standard and sample, indicative of stability by forced degradation test, linear (100-900 µg/mL), precise (RSD < 2%), accurate and rugged to modifications in the analytical process. The method was able to quantify commercial products, showing an average content of 98.95%. The greenness of the developed method presented NEMI with 4 green quadrants, ESA and AGREE with a score of 83 and 0.61, respectively, and GAPI predominantly green and yellow.

Conclusions: The method was selective, indicative of stability, linear, precise, accurate, rugged and green, by NEMI, ESA, AGREE and GAPI, to quantify IVE in injectable solution. Additionally, it combined the advantages of TLC and digital image analysis.

Highlights: The work shows a TLC method assisted by digital images for analysis of ivermectin-based product.

Background: Fodder crops are widely used as main ingredients of animal feed products. To combat pest infestations, farmers often apply pesticides on farms, the residual content of which may accumulate at or beyond toxic levels in/on the green fodder at the stage of harvest. To safeguard animals and humans (through ecological food chains) from these residual pesticides, both domestic and commercial programs are necessary to monitor the levels of pesticide residues in food and feed. Nonetheless, the existing methods exhibit constraints regarding scope, selectivity, and sensitivity. These limitations warranted a high-throughput, multiresidue method for monitoring and risk assessment of multiclass pesticides in fodder crops.

Objective: The study aimed to develop and validate a multiresidue method for the simultaneous analysis of 451 multiclass pesticides, their isomers, and metabolites of toxicological concern in three widely used fodder crops, namely sorghum, maize, and lucerne.

Methods: The well-homogenized samples of sorghum, maize, and lucerne (10 g) were extracted with acetonitrile (10 mL). An aliquot of the extract was cleaned by dispersive solid phase extraction (dSPE) with graphitized carbon black (GCB, 7.5 mg/mL). The method performance was evaluated for a mixture of multiclass pesticides at 10 µg/kg and 20 µg/kg using liquid and gas chromatography with tandem mass spectrometry (LC-MS/MS and GC-MS/MS).

Results: The GC-MS/MS and LC-MS/MS techniques allowed analyses of the test pesticides within chromatographic runtimes of 17 min and 20 min, respectively. The method's performance using matrix-matched calibration was satisfactory for all compounds (recoveries = 70-120%, repeatability-RSD, <20%) at 10 and 20 µg/kg in three studied matrices.

Conclusions: The method successfully determined the residues of all tested compounds in each fodder matrix. It demonstrates satisfactory selectivity, accuracy, and repeatability. Given all of these, it is recommended for regulatory and commercial testing purposes.

Highlights: A high-throughput residue analysis method targeting 451 compounds in sorghum, maize, and lucerne involved a single multiresidue extraction, followed by dSPE cleanup with analysis using LC-MS/MS and GC-MS/MS. The method sensitivity met the EU-MRLs, and performance complied with the SANTE/11312/2021's quality control criteria.

Background: The growing need to increase the efficiency of routine analyses in quality control laboratories of agricultural fertilizer manufacturers underscores the importance of automating official methods or developing validated alternatives to enhance analytical throughput. Phosphorus, a key nutrient in fertilizers, demands precise and rapid analytical techniques that comply with current regulatory requirements.

Objective: This study aimed to evaluate Flow Injection Analysis (FIA) methods with molecular absorption spectrophotometric detection-based on official protocols-and compare them to a more advanced technique, Microwave-Induced Plasma Optical Emission Spectrometry (MIP-OES), for the quantification of elemental phosphorus.

Methods: Three methodologies were assessed: molecular absorption spectrophotometry based on the formation of yellow vanadophosphomolybdate, the formation of molybdenum blue, and MIP-OES. These methods were applied to fertilizer samples extracted using water and neutral ammonium citrate (NAC), as prescribed by official procedures.

Results: FIA methods provided a sampling throughput of up to 120 analyses per hour, while MIP-OES enabled up to 240 analyses per hour. Both spectrophotometric methods had limits of quantification (LOQ) near 1.0 mg/L. However, the yellow vanadophosphomolybdate method showed better linearity, up to 40 mg/L, compared to 20 mg/L for the molybdenum blue method. MIP-OES exhibited the highest sensitivity (LOQ < 0.2 mg/L) and the broadest linear range. All methods showed acceptable accuracy and precision when tested with certified reference materials.

Conclusions: FIA systems are advantageous over conventional methods due to their simplicity and low cost, making them suitable for routine laboratory settings. MIP-OES, while requiring greater instrumental investment, offers higher throughput and eliminates the need for reagent preparation.

Highlights: Considering their respective strengths-FIA for accessibility and MIP-OES for performance-it is recommended that both methods are included as Official Methods of Analysis by the Brazilian Ministry of Agriculture (MAPA) and other international regulatory bodies.

Background: Molnupiravir, an FDA-approved antiviral for the treatment of COVID-19, requires reliable analytical methods to ensure its quality and safety due to its therapeutic importance.

Objectives: This study presents the development of a stability-indicating RP-HPLC method for estimating molnupiravir-related impurities in capsule formulations. An unknown impurity is structurally elucidated using LC-TQ/MS and 1H and 1³C NMR spectroscopy. Its potential safety profile is also assessed In-silico using the admetSAR tool.

Methods: The method demonstrated linearity (R2 ≥ 0.9990), precision (RSD < 1.34%), and sensitivity, with an LOD of 0.2 µg/mL and an LOQ of 0.4 µg/mL. Forced degradation revealed an unknown impurity at 2.4 min, which was structurally identified as N-hydroxycytidine using LC-MS and NMR. In-silico analysis indicates potential hepatotoxicity, mitochondrial toxicity, and reproductive toxicity.

Results: The method demonstrated linearity (R2 ≥ 0.9990), precision (RSD < 1.34%), and sensitivity, with an LOD of 0.2 µg/mL and an LOQ of 0.4 µg/mL. Forced degradation revealed an unknown impurity at 2.4 min, which was structurally identified as N-hydroxycytidine using LC-MS and NMR. In-silico analysis indicates potential hepatotoxicity, mitochondrial toxicity, and reproductive toxicity.

Conclusion: The validated RP-HPLC method successfully detects and quantifies molnupiravir impurities, supporting routine quality control and regulatory compliance. The unknown impurity's toxicity profile suggests the need for in vivo safety studies before official monograph inclusion.

Highlights: This research introduces a precise RP-HPLC method to evaluate molnupiravir's quality for COVID-19 treatment. It identified an impurity, characterized it, and predicted its safety to enhance our understanding of ensuring quality control for this vital drug.